(1) Why are you purifying your DNA. If you need to eliminate salt, then you can do this with drop dialysis, which is much easier and faster, and loses less sample.

I actually never got to know another method than microcolumn purification. Is the loss of DNA really so much smaller when using drop dialysis? And is drop dialysis feasible with a quite large ligation reaction (approx. 150 µl)?

(2) In my experience, ligations happen quickly (even with non- "quick ligase"). 5-15 minutes is all it takes, and can be done on the bench at room temperature.

For standard ligations, I also use quick ligase for 5 min or normal T4 ligase for 30 min.

I only do the overnight ligation for library-construction, where I use a lot of DNA (approx. 1.6 µg for vector+insert), because I was told that this is more reliable then...

(3) to answer your question, if I really wanted to keep a ligation over a weekend, I would do it by adding EDTA to chelate the magnesium, then store at +4, but why would you do that.

So to inhibit DNAse activity, I would add EDTA to which concentration?